Understanding of the biological roles for DNA repair, especially in relation to carcinogenesis and genotoxic cancer therapy will be pursued. DNA repair inhibitors will be studied in detail for mechanism, step, pathway, and cell organelle of action following damage to DNA by covalent attachment of drugs, and irradiation by x-ray and UV light. Particular attention will be directed toward cocarcinogenic agents shown in this laboratory to be repair inhibitors, and toward drugs which bind to DNA. Repair processes will be followed by isolating cellular DNA and monitoring the fate of thymine dimers and x-ray strand breaks in specific chromatin fractions both before and after replication of damaged cells. Drug combinations will be used to determine whether drugs act on identical or different steps, and drug analogs will be used to establish structure-function requirements for each class of drug inhibitor. Finally, individual "repair" enzymes will be tested for inhibitor effects when sites of action have been determined. Photoaffinity drug labeling will be used to produce covalent damage to DNA for study as a repair substrate. This technique developed in this laboratory, should permit attachment of radiolabeled drug to specific regions of the genome depending on the binding specificity of the drug used. This will provide a model for DNA damage which is much more relevant to drug carcinogenesis; will permit clear distinctions between nuclear and mitochondrial DNA damage and repair; and through use of radiolabeled drug should increase greatly the sensitivity of the detection of repair processes in animal cells. Detailed understanding of the mechanisms, specific steps, and subcellular sites of action of DNA damage and repair inhibitors will provide the means of establishing the biological roles for repair in both normal and diseased cells.